Mucin-type O-glycans are primary components of the colonic mucus gel layer. Altered intestinal O- glycosylation has been observed in patients with ulcerative colitis (UC), but whether this alteration is an etiological factor is unknown. O-glycans consist mainly of core 1- and core 3-derived O-glycans. The biosynthesis of these two types of O-glycans is controlled by core 11,3-galactosyltransferase (T-synthase) and Core 31,3-glucosaminyltransferase (C3GnT), respectively. T-synthase function requires a specific chaperone, Cosmc. Human Cosmc is on the X-chromosome, increasing the significance of somatic mutations in this gene. In preliminary studies, we detected somatic mutations in the Cosmc gene in DNA, isolated from colonic epithelial cells, expressing abnormal O-glycans from UC patients. We hypothesize that altered O-glycans impair mucus barrier function, which in turn allows intestinal microflora to interact abnormally with epithelium and mucosal immune cells, thus causing colitis. To test this hypothesis, we developed mice lacking either core 1- or combined core 1- and core 3-derived O-glycans (IEC T-syn-/- and DKO mice, respectively). In addition, we have also developed mice with tamoxifen (TM) inducible deletion of T-syn in intestinal epithelial cells (TM-IEC T-syn-/-). These mice develop spontaneous colitis, which is associated with a massive granulocyte infiltration and cryptic abscesses, closely resembling active human UC. Significantly, IEC T-syn-/- and DKO mice in the Rag1-/- background, who lack adaptive immunity, manifested similarly severe colitis, suggesting an essential role for innate immune cells such as granulocytes in colitis development. This supports an etiological role for O-glycans in colitis and provides a unique model system to test whether altered O-glycans is a potential molecular mechanism in the pathogenesis of human UC. We propose to 1) analyze how abnormal O-glycosylation impairs the expression of intestinal mucins and add additional patient samples to improve the statistical power of our preliminary observations that Cosmc mutations cause abnormal expression of colon epithelial O-glycans in UC patients; 2) determine the role of O- glycans in intestinal barrier function, investigate changes in bacterial variety or density in O-glycan-deficient mice before and after disease onset by phylogenetic analysis, and test definitively the role of microbiota in colitis development by developing germ-free O-glycan-deficient mice; and 3) identify mechanisms initiating granulocyte infiltration and determine the role of granulocytes in colitis. Our proposed studies will reveal novel insights into the pathogenesis of colitis and may lead to new therapies.